Page 64 - Chiral Separation Techniques

40 2 Method Development and Optimization of Enantiomeric Separations Using …

[27, 28]. Two chiral columns were also coupled to resolve three pairs of enantiomers
simultaneously [29].
Each glycopeptide CSP has unique selectivity as well as complementary charac-
teristics, and a considerable number of racemates have been resolved on all three of
them. Interestingly, most of the resolved enantiomers have the same retention order
on these macrocyclic CSPs. When they are mixed or coupled with each other, the
selectivity on one CSP will not be canceled by another. Even if some compounds
may not have the same retention order, the complementary effects will result in an
identifiable selectivity. Therefore, the coupled chiral columns can be used as a
screening tool and save chromatographers substantial time in method development.
In order to compare the retention and selectivity of coupled phases with individ-
ual CSPs, the parameters such as mobile phase composition, flow rate, detection
wavelength and operating temperature were maintained constant during the screen-
ing process. In the new polar organic mode, the mobile phase was methanol with
0.02 % glacial acetic acid and 0.01 % anhydrous triethylamine (v/v) and the flow
–1
rate 2 mL min . In reversed phase, the mobile phase was 25 % methanol and 75 %
–1
triethylammonium acetate buffer (0.1 %, pH 6) (v/v) and the flow rate 1 mL min .
In normal phase, the mobile phase was 40 % hexane and 60 % ethanol (v/v) and the
–1
flow rate 1.5 mL min . The minimum column length was determined to be 10 cm
to achieve efficient selectivity. The appropriate sequence of columns was important
to the coupling practice. It proved to be beneficial that ristocetin A column, which
usually has the lowest retaining ability, is put at the first position, whereas
teicoplanin – which usually has the highest retaining ability – is at the last position.
In the new polar organic mode, it was found that the capacity factors of the ana-
lytes on the coupled columns, i.e., ristocetin A plus teicoplanin (R+T), ristocetin A
plus vancomycin (R+V), and ristocetin A plus vancomycin plus teicoplanin
(R+V+T), is approximately the average of those on the individual columns [30].
Although there is not enough evidence to show a linear relationship between reten-
tion and the composition of a stationary phase, it is likely that each stationary phase
contributes equally to the retention. The contribution of each stationary phase to the
selectivity and resolution is more complicated. In some cases, where vancomycin
(V) and teicoplanin (T) had slightly different selectivity, the coupled columns (V+T)
showed selectivity as good or better than the two individual columns. For these com-
pounds, the coupled 10 cm columns (V+T) worked as well as a single 25 cm ana-
lytical column. However, for most compounds the coupled columns showed a
medium selectivity compared to the individual columns. The individual column with
the lower selectivity acts as a diluting factor. However, in the new polar organic
mode, the diluting effect from the column with lower selectivity is not very strong
(Fig. 2-7). Therefore, it is always beneficial to couple three columns in this mobile
phase in order to achieve the broadest selectivity in the shortest time.
One potential problem associated with column coupling in reversed phase is rel-
–1
atively high back-pressure (~ 2600 psi at 1 mL min ). This will place a limit on the
flow rate, which in turn limits the further reduction of analysis time. Also, compared
to the new polar organic mode, the retention in reversed phase on coupled columns
is deviated more from the average retention on the individual stationary phases,